1. Field of the Invention
The invention relates to compositions of matter capable of serving as residues for specific binding of third strands to double-stranded complementary nucleic acids of any base-pair sequence.
2. Description of Related Art
Definitions
Before describing related art, it may be useful to define certain terms to be used throughout the specification in describing the present invention:
Canonical basexe2x80x94any one of the standard nucleic acid bases, adenine-A, guanine-G, cytosine-C, thymine-T, uracil-U.
Canonical base pairxe2x80x94the complementary or Watson-Crick base pairs, AT/U and GC, formed from canonical bases.
Canonical base tripletxe2x80x94a base triplet formed by the interaction of a canonical third strand base and a canonical base pair.
Direct base pairxe2x80x94a target base pair with its purine base located in a homopurine strand of a target duplex sequence, i.e., AT/U, GC.
Inverted base pairxe2x80x94a target base pair with its pyrimidine base located in a purine-rich strand of a target duplex sequence, i.e., U/TA, CG. An inverted base pair therefore interrupts the continuity of a homopurine strand sequence.
Triplex motifxe2x80x94triplex stereochemistry as determined by the predominant bases of the third strand binding to a target Watson-Crick duplex with purine-rich, pyrimidine-rich strands and by the orientation of the third strand relative to that of the purine-rich strand of the target. Note that the mode of third strand base or residue H-bonding to the target base pair is characteristic of each motif.
N-residuexe2x80x94a synthetic third strand residue designed to bind with specificity to a particular direct target base pair or inverted target base pair.
Oligonucleotides (third strands) can bind to double-stranded nucleic acids to form triple-stranded helices (triplexes) in a sequence specific manner (Beal and Dervan, Science 251: 1360 (1991); Beal and Dervan, Nucleic Acids Res., 20:2773 (1992); Broitman and Fresco, Proc. Natl. Acad. Sci. USA, 84:5120 (1987); Fossella, et al., Nucleic. Acids Res. 21:4511 (1993); Letai et al., Biochemistry 27:9108 (1988); Sun, et al., Proc. Natl. Acad. Sci. USA 86:9198 (1989)).
The third strand binding code (a complementarity principle) dictates the sequence specificity for binding third strands in the major groove of double-stranded nucleic acids to form a triple-stranded helix or triplex. The code provides the specificity of third-strand binding for design of gene-based therapeutics that bind specifically to target nucleic acid sequences with little or no non-specific binding to non-target sequences.
Third-strand binding differs from the familiar Watson-Crick complementarity principle (A:T/U and G:C) for the double-stranded helix in two major respects: (1) the third-strand binding code is degenerate, and (2) third strands bind only to double-strands which contain a sequence of adjacent (or run of) purine bases (A or G) in one of the strands, which here will be called the center or core strand. The third-strand binding code is illustrated in the Table 1 below.
In the center of the table, a xe2x80x9c+xe2x80x9d means the bases are complementary or correspondent, and a xe2x80x9cxe2x88x92xe2x80x9d means they are not complementary or not correspondent. The bases are: A=adenine (purine); G=guanine (purine); C=cytosine (pyrimidine); T=thymine (pyrimidine); U=uracil (pyrimidine in RNA); I=inosine (purine nucleoside with the universal third-strand binding base hypoxanthine).
A serious practical limitation for stable third-stand binding dictated by the code in Table 1 is the necessity for runs of purines in the center target strand of typically 10 or more bases interrupted by only one or two pyrimidines (hereafter called xe2x80x9cpurine-richxe2x80x9d sequences or targets). While runs of sufficient length are present in many of the genes and the non-gene DNA (or RNA) of eukaryotes and prokaryotes and their viruses, they are not frequent enough for widespread diagnostic and therapeutic uses. It is therefore desirable to be able to target a duplex nucleic acid segment with a mixed purine and pyrimidine composition in the center strand.
There are a number of xe2x80x9cmotifsxe2x80x9d which further define third-strand binding to purine-rich targets, still in conformity with the third-strand binding code. The motifs define the base-compositional features of the third strand and whether the third-strand binds parallel or antiparallel to the purine-rich target strand (polarity). Motifs thereby define the hydrogen-bonding (H-bonding) schemes of the third-strand bases to the target base pairs. In consequence, the motifs also determine target specificity and nearest neighbor effects on binding. There are five motifs that describe third-strand binding (Sun and Helene, Current Opinion in Structural Biology. 3:345 (1993)). Table 2 summarizes the five motifs, which constitute a subset of constraints to the binding code. Thus, the motifs provide further instructions for defining the sequences of different third-strands that can alternatively bind with specificity to the same target. The Table also gives examples of selected analog bases which may be substituted for the standard or canonical A, G, T/U and C third-strand bases.
In the Binding Code column, the colon indicates third-strand binding of the base to the left of the colon, to at least the center purine base on the immediate right of the colon. The + superscript indicates that the base is in the protonated form when it binds (the energy of binding provides the energy for protonation). 2,6 DAP stands for 2,6 diaminopurine. xe2x80x9cParallelxe2x80x9d or xe2x80x9cantiparallelxe2x80x9d refers to the relation of third-strand polarity in the triplex relative to the purine-rich target strand.
In designing third strands, there are other considerations that can affect the stability of the resulting triplex. Third strands composed of A and G bases, for example, have the potential for several kinds of self structure. Very G-rich third strands tend to form either hairpin or linear helices stabilized by G-tetrads (Fresco and Massoulie, J. Am. Chem. Soc., 85:1352 (1963); Zimmerman, et al., J. Mol. Biol., 92:181 (1975); Gern, et al. Biochemistry, 34:2042 (1994)). With more equal portions of A and G bases, linear or hairpin duplexes with AG, AA and GG base-pairs form, as well as the tetraplexes, which have melting temperatures (Tm) in the same ranges as do dissociations of such third-strands from target duplex. Such third-strand self structures will obviously weaken third-strand binding by altering the equilibrium from triplex toward the duplex plus self-structured third strand. The processes and compositions of this invention can be utilized to devise third strands with reduced tendency for self-structure.
U.S. Pat. Nos. 5,034,506, 5,166,315 and 5,405,938 are directed to various polymers said to be effective to bind to Watson-Crick base pairs. In contrast to the present invention, however, those patents do not precisely model the stereochemistry of the canonical base triplets; nor do they precisely model the stereochemistry of their designed residues. Instead, they are directed to flexible non-native backbones that, upon triplex formation, are possibly capable of assuming locations acceptable for triplex formation. One concern with this approach is that backbones of greater flexibility than native sugar-phosphate backbones may suffer unacceptable negative entropy changes (positive free energy changes) when they are xe2x80x9cfrozenxe2x80x9d into the helical configuration demanded by triplex stereochemistry. This energetically unfavorable change may prevent stable triplex formation. In addition, those patents show residues only for the pyrimidine/parallel motif, not for the other four known triplex motifs.
WO 94/11534 is directed to third-strand residues which have been designed to bind to modified inverted duplex base pairs. The designed residues bind only to the core or center strand duplex base; and to do so, the center strand base must be modified to possess two hydrogen bonding sites. The necessity of duplex base modification to accomplish third-strand binding makes the invention of little use; in particular, it can have no use in therapeutics, since any disease target from a living organism would not possess the required modified bases that are the targets for such triplex formation.
The present invention relates to rules and guidelines for designing heterocycles and other structures, and to compositions of matter (xe2x80x9cresiduesxe2x80x9d) designed by those processes that when incorporated into third strands (with natural or synthetic backbones) make those strands capable of specific binding to complementary double-stranded nucleic acids of any base-pair sequence; that is, without the target requiring a purine-rich strand.
Accordingly, a major object of the present invention is to provide synthetic nucleic acid monomers (xe2x80x9cresiduesxe2x80x9d), that when incorporated into an oligonucleotide (xe2x80x9cthird strandxe2x80x9d), or analog oligomer, i.e., a third strand with a synthetic backbone, enables the third strand to form a triple-stranded nucleic acid (xe2x80x9ctriplexxe2x80x9d) when hybridized to a double-stranded nucleic acid (xe2x80x9cduplexxe2x80x9d), wherein the xe2x80x9ctarget regionxe2x80x9d to which the third strand binds is of substantially any base sequence; that is, it need not include a run of a large number of adjacent purines on one strand. In other words, the residues that are provided will be capable of strong and specific binding to inverted base pairs.
In one aspect, the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in a parallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising pyrimidine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 7.0 xc3x85 to about 8.6 xc3x85;
b) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone which is bound to the residue, is from about 53xc2x0 to about 82xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone, and the bond vector between the residue and the oligonucleotide backbone, is from about xe2x88x9290xc2x0 to about +90xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in a parallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 6.7 xc3x85 to about 8.6 xc3x85;
b) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone which is bound to the residue, is from about 81xc2x0 to about 125xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone, and the bond vector between the residue and the oligonucleotide backbone, is from about xe2x88x92100xc2x0 to about +60xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in an antiparallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 6.5 xc3x85 to about 8.6 xc3x85;
b) the e value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone which is bound to the residue, is from about 86xc2x0 to about 128xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone, and the bond vector between the residue and the oligonucleotide backbone, is from about xe2x88x9245xc2x0 to about +120xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in a parallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof, or 2,6-diaminopurine to bind to AT/U base pairs, and said residue(s) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 7.0 xc3x85 to about 8.6 xc3x85;
b) the xcex6 value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone which is bound to the residue, is from about 62xc2x0 to about 107xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone, and the bond vector between the residue and the oligonucleotide backbone, is from about xe2x88x9290xc2x0 to about +90xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a partially synthetic oligonucleotide containing a backbone having a polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide capable of binding in an antiparallel orientation relative to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof or 2,6-diaminopurine to bind to AT/U base pairs, and said residue(s) being substantially planar, such that when the oligonucleotide binds to the target sequence, base triplets are formed among each oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone which is linked to the corresponding residue of the oligonucleotide is from about 5.9 xc3x85 to about 8.2 xc3x85;
b) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone which is bound to the residue, is from about 90xc2x0 to about 110xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone, and the bond vector between the residue and the oligonucleotide backbone, is from about xe2x88x9230xc2x0 to about +120xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising pyrimidine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is linked to the corresponding residue of the oligonucleotide analog is from about 7.0 xc3x85 to about 8.6 xc3x85;
b) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue, is from about 53xc2x0 to about 82xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone, is from about xe2x88x9290xc2x0 to about +90xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is linked to the corresponding residue of the oligonucleotide analog is from about 6.7 xc3x85 to about 8.6 xc3x85;
b) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue, is from about 81xc2x0 to about 125xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone, is from about xe2x88x92100xc2x0 to about +60xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or residue and the corresponding bases of the target base pair of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is linked to the corresponding residue of the oligonucleotide analog is from about 6.5 xc3x85 to about 8.6 xc3x85;
b) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue, is from about 86xc2x0 to about 128xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone, is from about xe2x88x9245xc2x0 to about +120xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil or base analogs thereof or 2,6-diaminopurine to bind to AT/U base pairs, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is linked to the corresponding residue of the oligonucleotide analog is from about 7.0 xc3x85 to about 8.6 xc3x85;
b) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue, is from about 62xc2x0 to about 107xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone, is from about xe2x88x9290xc2x0 to about +90xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a substantially synthetic oligonucleotide analog containing a backbone having no polarity associated therewith, and nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said oligonucleotide analog being effective to bind in a sequence-specific manner to a target sequence of a duplex polynucleotide, said oligonucleotide analog capable of binding to a purine-rich or designated core or center strand of said duplex, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof or 2,6-diaminopurine to bind to AT/U base pairs, and said residue(s) being substantially planar, such that when the oligonucleotide analog binds to the target sequence, base triplets are formed among each analog oligonucleotide base or residue and the corresponding bases of the duplex, and each residue conforms to the following parameters:
a) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide analog backbone which is linked to the corresponding residue of the oligonucleotide analog is from about 5.9 xc3x85 to about 8.2 xc3x85;
b) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide analog backbone which is bound to the residue, is from about 90xc2x0 to about 110xc2x0;
c) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide analog backbone, and the bond vector between the residue and the oligonucleotide analog backbone, is from about xe2x88x9230xc2x0 to about +120xc2x0; and
d) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of:
a) providing a nucleic acid core or center strand which has a target sequence with 50% or more of purine bases;
b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand;
c) providing a third nucleic acid oligonucleotide or backbone analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising pyrimidine bases and/or base analogs thereof, and said residue(s) being substantially planar; and
d) contacting duplex formed from said core or center and complementary strands with said third strand, so as to allow formation of hydrogen bonds between the bases and residue(s) of the third strand and the target sequence of the core or center strand alone or together with the complementary strand, so as to form the triple-stranded nucleic acid, and each residue conforms to the following parameters:
i) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog which is linked to the corresponding residue of the oligonucleotide or backbone analog is from about 7.0 xc3x85 to about 8.6 xc3x85;
ii) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone or backbone analog which is bound to the residue, is from about 53xc2x0 to about 82xc2x0;
iii) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone or backbone analog, and the bond vector between the residue and the oligonucleotide backbone or backbone analog, is from about xe2x88x9290xc2x0 to about +90xc2x0; and
iv) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of:
a) providing a nucleic acid core or center strand which has a target sequence with 50% or more of purine bases;
b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand;
c) providing a third nucleic acid oligonucleotide or backbone analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar; and
d) contacting duplex formed from said core or center and complementary strands with said third strand, so as to allow formation of hydrogen bonds between the bases and residue(s) of the third strand and the target sequence of the core or center strand alone or together with the complementary strand, so as to form the triple-stranded nucleic acid, and each residue conforms to the following parameters:
i) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog which is linked to the corresponding residue of the oligonucleotide or backbone analog is from about 6.7 xc3x85 to about 8.6 xc3x85;
ii) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone or backbone analog which is bound to the residue, is from about 81xc2x0 to about 125xc2x0;
iii) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone or backbone analog, and the bond vector between the residue and the oligonucleotide backbone or backbone analog, is from about xe2x88x92100xc2x0 to about +60xc2x0; and
iv) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of:
a) providing a nucleic acid core or center strand which has a target sequence with 50% or more of purine bases;
b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand;
c) providing a third nucleic acid oligonucleotide or oligonucleotide analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising purine bases and/or base analogs thereof, and said residue(s) being substantially planar; and
d) contacting duplex formed from said core or center and complementary strands with said third strand, so as to allow formation of hydrogen bonds between the bases and residue(s) of the third strand and the target sequence of the core or center strand alone or together with the complementary strand, so as to form the triple-stranded nucleic acid, and each residue conforms to the following parameters:
i) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog which is linked to the corresponding residue of the oligonucleotide or backbone analog is from about 6.5 xc3x85 to about 8.6 xc3x85;
ii) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone or backbone analog which is bound to the residue, is from about 86xc2x0 to about 128xc2x0;
iii) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone or backbone analog, and the bond vector between the residue and the oligonucleotide backbone or backbone analog, is from about xe2x88x9245xc2x0 to about +120xc2x0; and
iv) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of:
a) providing a nucleic acid core or center strand which has a target sequence with 50% or more of purine bases;
b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand;
c) providing a third nucleic acid oligonucleotide or oligonucleotide analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof, or 2,6-diaminopurine to bind to AT/U base pairs, and said residue(s) being substantially planar; and
d) contacting duplex formed from said core or center and complementary strands with said third strand, so as to allow formation of hydrogen bonds between the bases and residue(s) of the third strand and the target sequence of the core or center strand alone or together with the complementary strand, so as to form the triple-stranded nucleic acid, and each residue conforms to the following parameters:
i) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog which is linked to the corresponding residue of the oligonucleotide or backbone analogis from about 7.0 xc3x85 to about 8.6 xc3x85;
ii) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide backbone or backbone analog which is bound to the residue, is from about 62xc2x0 to about 107xc2x0;
iii) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide backbone or backbone analog, and the bond vector between the residue and the oligonucleotide backbone or backbone analog, is from about xe2x88x9290xc2x0 to about +90xc2x0; and
iv) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.
In another aspect, the present invention provides a method of forming a triple-stranded nucleic acid comprising the steps of:
a) providing a nucleic acid core or center strand which has a target sequence with 50% or more of purine bases;
b) providing a complementary nucleic acid strand which is hydrogen bonded in a Watson-Crick manner to said target sequence on the core or center strand;
c) providing a third nucleic acid oligonucleotide or oligonucleotide analog strand comprising a natural or synthetic backbone, in the latter case that is directional or nondirectional, containing nucleotide bases and at least one synthetic residue bound to the backbone, the bases and residue(s) of said third strand being effective to bind in a sequence-specific manner to the target sequence, said nucleotide bases comprising guanine to bind to GC base pairs, and thymine or uracil and/or base analogs thereof or 2,6-diaminopurine to bind to AT/U base pairs, and said residue being substantially planar; and
d) contacting duplex formed from said core or center and complementary strands with said third strand, so as to allow formation of hydrogen bonds between the bases and residue of the third strand and the target sequence of the core or center strand alone or together with the complementary strand, so as to form the triple-stranded nucleic acid, and each residue conforms to the following parameters:
i) the radius of the imaginary circle connecting the C1xe2x80x2 ends of the two glycosyl bonds of the target base pair of the duplex polynucleotide and the atom of the oligonucleotide backbone or backbone analog, which is linked to the corresponding residue of the oligonucleotide or backbone analog is from about 5.9 xc3x85 to about 8.2 xc3x85;
ii) the "THgr" value, measured from the C1xe2x80x2 atom bound to the base in the core or center strand, to the atom of the oligonucleotide or backbone analog which is bound to the residue, is from about 90xc2x0 to about 110xc2x0;
iii) the xcex6 value indicating the angle between said imaginary circle radius passing through the atom of the residue which is bound to the oligonucleotide or backbone analog, and the bond vector between the residue and the oligonucleotide or backbone analog, is from about xe2x88x9230xc2x0 to about +120xc2x0; and
iv) the residue forms a total of at least two hydrogen bonds with one or both bases of the corresponding target base pair of the duplex.